PROJECT SUMMARY Histone demethylases, a class of chromatin-modifying enzymes, regulate gene expression by modulating the methylation status of histone proteins within chromatin. Demethylases that belong to the KDM5 family antagonize tri-, di-, and monomethylation of lysine 4 in the tail of histone H3. In humans, this family has four members, KDM5A-D. While in physiology these enzymes act as regulators of development and differentiation, their misregulation via overexpression or mutagenesis is causative of cancer (KDM5A and B) and intellectual disability (KDM5C). Despite their importance, the mechanisms by which the catalytic activities of these enzymes are regulated on chromatin are still unknown. In addition to the catalytic domain, these demethylases contain several other domains, including two to three chromatin ?reader? domains belonging to the plant homeodomain (PHD) family. Prior to our work, PHD domains in demethylases and demethylation complexes had been shown to act as chromatin binding modules to regulate recruitment and substrate specificity of demethylases. By investigating regulation of catalysis in demethylase KDM5A, we uncovered a novel function of PHD domains in demethylases. Specifically, we demonstrate that occupancy of the PHD1 domain by a ligand peptide allosterically stimulates demethylation activity of KDM5A. The preferred ligand for the PHD1 domain is unmodified histone H3 peptide (H3K4Me0), and the binding affinity of this domain for histone H3 peptide progressively decreases with the increase in methylation of Lys 4. Our work demonstrates that PHD domains can actively regulate catalytic activity of KDM5 demethylases and defines a new way in which the function of reader and catalytic domains are coupled to regulate demethylation. As the allosteric stimulation exploits a positive feedback-based mechanism, our findings suggest a model by which demethylation could spread on chromatin. The objective of this application is to define the role of allosteric regulation in controlling the catalytic activities of KDM5 demethylases. In Aim 1, we will determine the molecular basis of histone tail recognition by the PHD1 chromatin reader domain of KDM5A and quantitate the impact of post-translational modifications on recognition. In Aim 2, by interrogating how ligand binding to the PHD1 domain influences demethylation in the context of catalysis-competent constructs, we will determine the impact of the PHD1 domain occupancy on the catalytic activity of KDM5 demethylases. In Aim 3, by using in vitro reconstituted chromatin substrates, we will evaluate whether the functional coupling between the PHD1 domain and the catalytic domain can allow for spreading of demethylation on chromatin. By providing mechanistic insight into the functional coupling between the reader and catalytic domains in KDM5 demethylases, our approach will expand our understanding of the mechanisms that underlie regulation of chromatin methylation and consequently transcription.